Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Eukaryotic TLS Pols display a high degree of specialization in their proficiency for replicating through different types of DNA lesions. Among these Pol? is unique in its proficient ability for error-free replication though UV induced cyclobutane pyrimidine dimers (CPDs), and inactivation of Pol? in humans causes the cancer- prone syndrome, the variant form of xeroderma pigmentosum (XPV). Pol? also promotes proficient and error-free replication through DNA lesions induced by chemical carcinogens and DNA adducts formed from the action of reactive oxygen species. To elucidate the role of Pol? in lesion bypass, we will use a combined biochemical, genetic, and structural approach to determine how Pol? mediates proficient and error-free replication through DNA lesions such as a UV induced cis-syn TT dimer, an 8-oxoguanine formed from oxidative damage, and a cisplatin G-G crosslink formed upon cisplatin chemotherapy. In Aim 1, we will determine Pol?'s mechanism of nucleotide incorporation by a comparison of binary and ternary structures with undamaged DNAs. The structures will be tested by mutagenesis and kinetic studies aimed at defining the reaction pathway of this unique polymerase. In Aim 2, we will determine structures of Pol? in ternary complex with DNAs containing a cis-syn T-T dimer, a (6-4) TT photoproduct, an 8-oxoguanine, and a cisplatin G-G intrastrand crosslink. We hypothesize that Pol? differs from classical as well as other TLS polymerases in the openness of its active site cleft, enabling it, for example, to accommodate both Ts of the T-T dimer. The structures will tested by biochemical and genetic methods aimed at defining Pol?'s action on DNA lesions. In Aim 3, using biophysical and biochemical approaches, we will test the hypothesis that even though most of the missense mutations in XP-V patients lie distant from the active site, they adversely affect Pol? structure and function because of their effects on the structural stabilities of Pol? domains. Together, the studies we propose here will make an important contribution toward delineating the mechanisms by which Pol? guards cells against the genotoxic effects of sunlight-induced chromosomal damage as well as damage induced from endogenous oxidation reactions and by chemotherapy. The proficient ability of Pol? for mediating error-free replication through DNA lesions formed from exposure to environmental and chemical carcinogens and from cellular oxidative reactions will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The proposed studies are highly relevant for cancer biology and etiology, as they will reveal how Pol? minimizes the mutagenic and carcinogenic potential of DNA lesions in human cells.